Figure 7: SFK activity is necessary for TLR-ligand localization in early endosomes of pDCs.

(a) BM-derived DCs cells were pre-treated for 1 h with PP2 (10 μM; blue) or DMSO control (black) before stimulation with either CpG-A-FITC or CpG-B-FITC for 1 h. CpG uptake was determined by flow cytometry in gated cDCs and pDCs. pDCs were gated as CD11c⁺/CD11b⁻/BST2⁺. Data are representative of three independent experiments. Graphs depict mean±s.d. of replicates within one representative experiment. (b,c) BM-derived DCs were pre-treated for 1 h with PP2 (10 μM; blue) or DMSO control (black), before stimulation with CpG-A-FITC for 90 min. CpG-A-FITC internalization and colocalization with EEA1 or LAMP2 was evaluated by confocal microscopy in B220⁺ (b, pDCs) and B220⁻ (c, cDCs) cells. Examples of representative images are shown, with RGB plots underneath each panel. These plots quantify the degree of colocalization of CpG-A-FITC (green) with EEA1/LAMP2 (red) along the arbitrary line on each corresponding image, as measured using ImageJ. Bar graphs display the patterns of CpG-A-FITC localization/uptake and % colocalization with EEA1 determined in 50 cells analysed in two independent experiments (b). CpG uptake in cDCs is graphed as percentage of positive cells within 50 cells analysed in two independent experiments (c). Two-ways Student’s t-test (a,b) and χ²-test (b,c) were used for statistical analyses. Scale bars, 10 μm. *P<0.05, **P<0.01, ***P<0.001.